While the precise roles of most replicase gene proteins in SARS-CoV replication and pathogenesis are not known, available data suggest that they likely play critical roles in replication in culture and in replication and pathogenesis in animals. The overall goal of this project is to define the requirements for SARS-CoV replicase protein expression and processing for virus growth in culture and for replication, immune response, and protection in animals. Genetic approaches will be used to introduce mutations in the replicase gene that will alter protein processing, inactivate putative functional proteins, and determine the capacity for chimeric protein function and virus recombination. The effects of these changes will be determined in culture by assessing virus growth, protein expression and processing, and RNA synthesis, and in animals by assessing replication, immune response, and protection. The determination of replicase requirements and the development of replicase mutants will serve as the basis for the development of mutant viruses to be grown safely for inactivation or for further study as candidate live-attenuated live virus vaccines. The Specific Aims of the program are: 1) to determine the role for replicase polyprotein cleavage events and processing intermediates in SARS-CoV replication; 2) to determine the requirement for putative RNA processing enzymes (nspl4, 15, and 16) in viral replication; 3) to determine the ability of SARS-CoV to function with chimeric replicase proteins and to recombine with MHV; and 4) to evaluate the replication and immune response from viable replicase mutants and the protection from wildtype SARS-CoV challenge in mice and ferrets.